We developed a frequency-domain fluorometer which operates from 4 to 2000 MHz. The modulated excitation is provided by the harmonic content of a laser pulse train (7.59 MHz, 5 psec) from a syncronously pumped and cavity dumped dye laser. The phase angle and modulation of the emission are measured with a microchannel plate photomultiplier. Cross-correlation detection is performed outside the PMT. The performance was verified by measurement of known time delays and examination of standard fluorophores. The detector displayed no detectable color effect, with the 300 to 600 nm difference being less than 5 psec. The precision of the measurements is adequate to detect differences of 20 psec for decay times of 500 ps.

A simple method of determining the errors in a multicomponent analysis by phase resolved luminescence spectroscopy (PRS) is presented. Without resorting to many tedious experiments, one can determine a priori the optimum phase angles and frequencies for an analysis based only on the component's lifetimes. The approach allows estimation of the theoretical limiting precision of a data acquisition-reduction method. This, in turn, allows assessment of whether different noise sources or systematic errors are present and where improvements in experimental design can enhance the measurement. Finally, it allows direct analysis of the effect of different types of noise sources. An evaluation of the errors in a two-component analysis in the presence of photon noise is presented.

This paper describes a system capable of measuring fluorescent lifetimes and time-resolved spectra of microscopic particles, thus substantially enhancing the usefulness of fluorescence microscopy as a characterizing tool. Tests and applications of this technique for identifying component lifetimes (in the nanosecond and subnanosecond range) and spectra in organic model compounds also are described. A multi-exponential decay fit to the data yields fluorescence decay times, percentage contributions (fractional intensities) and time-resolved spectra for each decay. Two-component non-interacting mixtures are resolved if the decay times differ by a factor of two and the percentage contribution from each fluorophore is more than 10%. For three-component mixtures, it is necessary for the resolution of the lifetimes that the decay times differ by at least a factor of three and the percentage contribution from each component be more than 15%.

The utility of thermally modulated emission (TME) spectroscopy as a technique for elucidating the properties of the excited states of molecules and complexes with near-degenerate excited electronic states is reviewed. A description of the TME method, its realm of application, and the type of information obtained are given. Particular emphasis is placed on the use of infrared heating as a tool for observing spectra in diverse media.

The electric field modulation of time resolved fluorescence has been studied in a N,N' bis(methyl)perylene 3,4,9,10 tetracarboxyl diimide. It was found that fluorescence is multiphasic in nature which was interpreted in terms of energy transfer from host molecules to defect sites. Analysis of the experimental data showed that the application of the electric field leads to an increase in the decay rate of fluorescence but leaves the initial fluorescence intensity unchanged. These results indicate that true intrinsic carrier generation process exists which occurs by electric field assisted dissociation of the first excited singlet state.

We are developing a novel scheme for ultrasensitive isotopic analysis of noble gases which is based on photon-burst detection of the 1s⁵ metastable state. This scheme and recent progress are discussed.

A method has been developed for making time-resolved measurements of the ruby fluorescence spectrum under shock compression. Preliminary experiments to demonstrate the feasibility of this technique are presented and recent modifications of the apparatus to provide quantitative data are summarized. Future experiments to determine the rate of change of wavelength with stress, as well as the effects of temperature and crystal orientation, are indicated.

Monocrystals of nickel, copper, germanium and germanium doped with copper mounted to a Bragg spectrometer are excited to produce K-hole states irradiated by a collimated beam of ionizing radiation of L-series photons and bremsstrahlung from a tungsten target. Excited monocrystals emit directed K-series fluorescence as Kossel photons. The observation of an unusual abrupt increase in intensity of K-series Kossel photons as the crystal is tuned to have the Bragg resonance angle with respect to the collimated ionizing beam of tungsten radiation is essentially the primary finding in this presentation. An amplification has been observed by using a flash x-ray source to produce a high rate of K-hole state in the lasing crystal and when a directed strong beam of K^α_l photon at Bragg resonance angle is channeled through the lasing crystal. This observation of monochromatic, coherent Kossel and nearly nondivergent K^α_l photons from an excited monocrystal pumped by a collimated beam of radiation leads to the design of a novel portable sealed off x-ray tube for investigation of small angle scattering, x-ray diffraction of polymers, ceramics, composites, biological samples and other materials of technical importance.

A series of fluorescence probes (p-(N,N-dialkylamino) benzylidene malononitriles) which belong to a class of organic compounds known as "molecular rotors" has been used to study solid polymer films. The internal molecular rotation of these compounds can be slowed down by increasing the surrounding media rigidity, viscosity or decreasing the free-volume available for molecular relaxation. Inhibition of internal molecular rotation of the probe leads to a decrease in the non-radiative decay rate and consequently enhancement of fluorescence. This behavior can be used to study both the static and dynamic changes in free-volume of polymers as a function of molecular weight, stereoregularity, crosslinking, polymer chain relaxation and flexibility. In addition, the dependence of the fluorescence emission maximum of these probes on media polarity allow continuous monitoring of the probes location in the polymer matrix. These fluorescence materials are capable of simultaneously probing the flexibility and polarity of their surrounding media.

A novel approach to investigation of electrical damage in insulators is described. It involves the use of fluorescence probes that selectively stain the damage features of interest or that react at specific sites, changing their fluorescence characteristics. Because of the sensitivity laser excited fluorescence provides, surface flashover tracks, locations of corona discharge and other features are easily revealed even when no damage is discernible in room light. Our results show that structural features, charge distributions, and chemical changes caused by electrical damage may be probed. Some surprising features of insulator damage revealed by fluorescence probes are presented.

When materials are deformed and broken, surfaces are formed which may have charge concentrations as well as defects and displaced atoms. The consequences of such departures from non-equilibrium can lead to the emission of particles (electrons, ions, and neutral species) as well as photons (triboluminescence). Collectively, we refer to these emissions as fracto-emission. We present measurements of various components of emission from a variety of materials and show that a number of features of the emission involve the transport of charge.

Photoluminescence spectroscopy is one of the most effective methods for characterizing semiconductor materials like silicon, gallium arsenide or indium phosphide. This analytical technique supplies data that tells the researcher how well devices fabricated from a. particular material can be expected to perform. The information can then be used to optimize growing conditions or to reject a sample before additional processing. Such increases in, and stabilization of device yield are critical in a very competitive marketing environment.

A fast analog technique developed for the determination of fluorescence emission parameters of microscopic geological particles is applied to a variety of samples. Presented here are fluorescence data from liptinite coal macerals, analysis of fluorescence emitted from crude oils, applications to fiber analysis and plastic scintillators. Characteristic decay times in the nanosecond and subnanosecond range are given along with time-resolved emission spectra.

Characteristic K-series, L-series, etc. x-ray photons are produced when the atoms are excited to ionize a K-shell electron of the atom. The generation of such a K-hole state followed by K-, L-series emissions from the element unambiguously identity the element. The initial K-hole state of excitation can be done either by electron bombardment or photon bombardment. The results presented here are due to photon bombardment. The simplicity of the instrument and its application to know the presence of trace elements in geological and biological samples of normal and abnormal tissues is an attractive feature.

A porous membrane "Diffusion Scrubber" represents an ideal interface between trace atmospheric gases of interest and sensitive fluorescence-based continuous liquid phase analysis methods for the corresponding analytes. Analytical methods capable of determining low to sub-parts-per-billion levels of hydrogen peroxide, sulfur dioxide, ammonia and formaldehyde on a continuous basis are described.

The dynamical behavior of large molecules can be studied by measuring the fluorescence of fluorophores bound to the molecule. Time-resolved fluorescence, using frequency-domain technique, provides a unique tool for the analysis of the emission from complex systems. Time resolution of few picoseconds is attainable using state-of-the-art commercial phase and modulation fluorometers. The development of these high-resolution instruments allowed for a new understanding of the internal motions of proteins and of molecules embedded in natural and synthetic membranes.

Measurements of time-resolved fluorescence are often used for studies of biological macromolecules. Such measurements are usually performed in the time-domain, by measurement of the time-dependent emission following pulsed excitation. It has recently become possible to measure the frequency-response of the emission to intensity modulated light, over a wide range of modulation frequencies. We used frequency-domain fluorometers which operates from 1 to 220 MHz, and more recently to 2000 MHz. The frequency-domain data provide excellent resolution of time-dependent spectral parameters. It is now possible to resolve closely spaced fluorescence lifetimes, to determine multi-exponential decays of anisotropy and to determine time-resolved emission spectra of samples which display time-dependent spectral shifts. In this article we show representative results on tryptophan fluorescence from proteins and for protein-bound fluorophores.

Distributions of fluorescence lifetimes which underly a fluorescence decay curve may be recovered by a straightforward analytical procedure. The procedure is model independent and requires no previous knowledge of the underlying distribution. The procedure has been tested on a variety of simulated and real data and applied to real experimental systems.

Nanosecond pulse fluorescent spectroscopy is a very powerful method for studying protein structure in solution. Studies using this methodology fall into two main categories, measurement of the decay of total fluorescence to evaluate fluorescence lifetime, and time-resolved fluorescence anisotropy measurements of the decay of polarization. The experiments are performed by attaching fluorescent probes to various known sites on protein molecules. The lifetime experiments tell us about the dynamic properties of the fluorescent moiety itself, while the anisotropy measurements provide information about the shape and hydrodynamic properties of the protein to which the fluorescent moiety is attached. Fluorescent probes that show only simple fluorescence can be used for studies with lifetimes up to about 120 ns; probes that form triplets will have longer lifetimes into the microsecond or even millisecond range.

We are using time-dependent and steady state measurements of fluorescence to study the physical interaction between DNA and carcinogenic hydrocarbons. The (±)7,8-diol-benzo[a]pyrene(7,8-diolBaP) is used as a model compound that forms a physical complex with DNA by intercalating between the DNA base pairs. An exact emission spectrum can be obtained by measuring the decay-associated emission spectra. In this technique the time-dependent fluorescence decay is measured using time-correlated single-photon counting detection. The data is fit to a sum of exponentials using a weighted non-linear regression program to obtain amplitudes and lifetimes of the emitting species at various wavelengths. Plotting the normalized intensities of a particular lifetime as a function of wavelength yields the emission spectrum associated with the lifetime. The measured fluorescence lifetime is 26.7 nsec for the free 7,8-diolBaP in solution and 5.0 nsec for the bound 7,8-diolBaP. The time resolved emission spectrum of bound hydrocarbon is red-shifted by 6 nm, characteristic of an intercalated complex. Fluorescence quenching experiments with iodide, an external quencher of DNA, gave a bimolecular dynamic quenching constant, 1.4 (10)⁹ (Msec)^-1, for the free hydrocarbon and a constant lifetime and amplitude for the bound 7,8-diolBaP. Fluorescence quenching with low concentrations of silver ions, which bind predominantly to guanine sites of DNA, results in reduced fluorescence intensity of the bound hydrocarbon, with the lifetime remaining unchanged. These quenching studies indicate that at least part of the fluorescence emitted is from 7,8-diolBaP bound at guanine sites. In addition, results of quantum yield experiments have been used to calculate the fraction of bound hydrocarbon which is totally quenched (52%) and not totally quenched (25%). The remainder is free in solution. Quenching experiments with mercury ions suggest that the totally quenched hydrocarbon is bound at adenine-containing sites.

Lipids and proteins are important functional and structural components of living organisms. Although proteins are frequently found as soluble components of plasma or the cell cytoplasm, many lipids are much less soluble and separate into complex assemblies that usually contain proteins. Cell membranes and plasma lipoproteins' are two important macro-molecular assemblies that contain both lipids and proteins. Cell membranes are composed of a variety of lipids and proteins that form an insoluble bilayer array that has relatively little curvature over distances of several nm. Plasma lipoproteins are different in that they are much smaller, water-soluble, and have highly curved surfaces. A model of a high density lipoprotein (HDL) is shown in Figure 1. This model (d - 10 nm) contains a surface of polar lipids and proteins that surrounds a small core of insoluble lipids, mostly triglycerides and cholesteryl esters. The low density (LDL) (d - 25 nm) and very low density (VLDL) (d 90 nm) lipoproteins have similar architectures, except the former has a cholesteryl ester core and the latter a core that is almost exclusively triglyceride (Figure 1). The surface proteins of HDL are amphiphilic and water soluble; the single protein of LDL is insoluble, whereas VLDL contains both soluble and insoluble proteins. The primary structures of all of these proteins are known.

The time decay of fluorescence intensity and anisotropy has been measured for an N-acetylaminoethyl-5-naphthylamine-l-sulfonate probe attached to Cys-98 of troponin C, a dansyl aziridine label linked to Met-25 of troponin C, and 2-p-toluidinyl naphthalene-6- sulfonate bound by calmodulin. In all cases the time decay of intensity at 25° was multi-exponential. The time decay of fluorescence anisotropy was also multi-exponential and could be fitted in terms of a long correlation time reflecting the rotational motion of all, or a major portion of the molecule, and a short correlation time arising from a more localized motion. For the Ca²⁺-liganded forms of both proteins the magnitude of the longer correlation time could be accounted for in terms of the structure of native calmodulin under these conditions. In the case of the label linked to Cys-98 of troponin C in the absence of Ca²⁺, the probe senses the rotation of a subelement of the molecule at 25°; at 40° the probe rotates almost independently of the overall structure.

A review of the advances in optical microscopy and the recent applications in biomedical research are discussed.

We have investigated the potential of lasers for real time in situ dental diagnosis via transillumination of teeth and gums and via fluorescence. Not surprisingly, absorption and/or scattering of light by teeth was found to be insensitive to light color. However, monochromatic transillumination revealed detail better than white light. Transillumination of gums was best performed with orange-red light because of tissue absorption. Illumination of oral structures by 488 nm Ar-laser light was effective in revealing diagnosis detail by fluorescence. Incipient caries and fine tooth fracture lines that are generally not revealed by radiography were observable by laser.

A recent application of fluorescence that is perhaps largely unknown outside law enforcement involves the utilization of laser excited fluorescence (LEF) in forensic science. In this overview, the focus is on LEF applicaton to development of latent fingerprints. Other areas of criminalistics, such as document examination and fiber analysis will be dealt with briefly only. To bring the technical reader, who likely has little familiarity with the fingerprint field, up to stream, a historical introduction precedes the description of current procedures for laser detection of latent fingerprints which is followed by a brief outline of current areas of research in the fingerprint area and application of lasers to other types of evidence examination. The overview concludes with an assessment of the current state and utilization growth prognosis of lasers in criminalistics.

Some of the advantages of copper vapor lasers (CVL) over argon ion lasers, from forensic scientist's viewpoint are being demonstrated. Of particular interest are the additional line at 578 manometer and the pulsed nature of CVI. Research projects involve the design of better chemical fluorescors for latent fingerprint detection and sensitivity enhancement by optronic means.

The powerful monochromatic radiation from argon ion lasers will excite fluorescence in small traces of many substances. This has proved to be of particular value in the detection and enhancement of latent fingerprints and other trace contact evidence. The photography of these marks is extremely important and can be difficult. Many specialised photographic techniques are used including both interference filters and sharp cut off filters, various photographic emulsions and infra red sensitive film. Hypersensitisation of the film and cooling in liquid nitrogen are used to improve the photography of weak fluorescence, various chemical treatments are also used to further enhance the fluorescence.

We have assessed the ability of laser-excited fluorescence to distinguish between similar inks, as might be found in document alterations. We find that the combined use of a coating procedure that strongly increases fluorescence efficiency at room temperature and fluorescence examination at 77°K provide high discriminatory capability. The procedures are easy to use and satisfy practicality requirements essential for utilization in document sections of law enforcement agencies.

We have surveyed the current methods for detection of blood prints and have compared representative procedures from the standpoint of the general type of involved chemistry. We find that the methods that involve fluorescent products produce the greatest sensitivity in concert with laser fluorescence excitation. The ninhydrin/ZnCl₂ procedure is very effective for porous items, including cloth. Merbromin or dichlorofluorescein are effective for non-porous surfaces. Development of blood prints on surfaces that display overwhelming background fluorescence is best performed by peroxidase-type absorption methods.

Two fluorescent probes, ANS and TNS, were found to be effective in detecting bloodstain patterns. These materials, which are not fluorescent in aqueous solution, react non-specifically with protein and enable an observer to discern a pattern when viewed under UV light at 366 nm.

A review of some basic image processing techniques for enhancement and restoration of images is given. Both digital and optical approaches are discussed. Fingerprint images are used as examples to illustrate the various processing techniques and their potential applications in criminalistics.

Laser detection of latent fingerprints tends to fail when prints are located on certain types of surface, such as brown cardboard and wood, because the generally weak fingerprint fluorescence is overwhelmed by background luminescence from these substrates. The fingerprint and background fluorescence lifetimes, which are of nanosecond order, were measured for samples of the above surface types and for fingerprints treated by the most successful current detection procedures. Our results indicate that time-resolved imaging can in these situations improve image contrast by about an order of magnitude.